CN109966484A - A kind of immunopotentiator, preparation method, avian influenza vaccine and application - Google Patents
A kind of immunopotentiator, preparation method, avian influenza vaccine and application Download PDFInfo
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Abstract
The invention belongs to veterinary biologics technical fields, disclose a kind of immunopotentiator, including Cys-Lys-Asp-Val-Tyr and astragalus polyose；The weight ratio of the Cys-Lys-Asp-Val-Tyr and astragalus polyose is 1:5~1:20；The immunopotentiator can effectively improve the antibody titer expression of vaccine immunity.And immunopotentiator preparation is simple, and prospects for commercial application is good, meanwhile, the invention also discloses the preparation method of the immunopotentiator, using the avian influenza vaccine of the immunopotentiator and the application in terms of avian influenza vaccine.
Description
Technical field
The present invention relates to veterinary biologics technical fields, in particular to immunopotentiator, preparation method, avian influenza vaccine
And application.
Background technique
Bird flu is the important communicable disease as caused by avian influenza virus, seriously endangers the development of poultry husbandry.Vaccine is exempted from
Epidemic disease is one of control most effective means of epidemic disease.But with the popularization and use of vaccine, virus is continuous under the selection pressure of vaccine
It morphs, virulence enhancing brings challenge to the prevention and control of epidemic disease.The solution above predicament, clinically constantly to replace vaccine
Strain reply.For the preventing and controlling of bird flu, the replacement of vaccine strain can have the effect got instant result really in a short time,
But on long terms, this also accelerates avian influenza virus speed of mutation, and prevention and treatment falls into the predicament of circulation.
Body immune system function can be improved in immunopotentiator, and enhancing body selects disease non-specific immunity
Suitable immunopotentiator and vaccine compatible use are to enhance one of the effective way of existing vaccine immunity effect.Vaccine is improved to exempt from
Effect, it is meant that less vaccine can but have a better effect, and clinical use can reduce immune time, reduction stress, both saved
Cost, and can be effectively controlled disease.
Bursin (BP) is the small molecule skin substance being present in bird bursal, has substantial connection with immune function.Capsule
Plain pentapeptide (BP5) is one of Bursin family, has induction poultry bone-marrow-derived lymphocyte precursor differentiation, proliferation, improves antibody water
It is flat, enhance humoral immunity；The activity of conversion for promoting T lymphocyte, to enhance the effect of cellular immunity.
Two more representative polypeptides are BP-14 and BP-5 in Bursin family.
About BP-14, obligee Jiangsu Agri-Animal Husbandry Vocational College applied for Chinese patent in 2013
CN201310413608.2 discloses a kind of polypeptide, the amino acid sequence of the polypeptide are as follows:
COOH-G-H-K-T-R-N-D-P-L-K-G-A-V-D-NH2。
Its effect is historically are as follows: by immunopotentiator BP14, uses various dose addition poultry according to different target animal
Simultaneous inoculation is carried out in epiornitic seedling；The antibody level of animal after immune is higher than vaccine group；Animal subject generates higher level
Cell factor.The dosage of fowl is 0.1 μ g/ plumage part, and pig is g/ parts of 5 μ；With reference to its Fig. 1, using BP-14+ vaccine (H5N1
Hypotype, Re-6 plants), titre 5-8log2.
About BP-5, obligee Li Deyuan applied for that Chinese patent CN200810243575.0 discloses one kind in 2008
Bursa pentapeptide, amino acid structure sequence are as follows: CYS-LYS-ASP-VAL-TYR.Bursa pentapeptide (BP5) structure of the invention
Simply, molecular weight is small, no chemical toxicity, non-immunogenicity, and preparation is simple, can both mention from the bursa of farbricius of chicken or other birds
It takes, can also be with chemical synthesis, cost is very low when chemical synthesis, can largely prepare.Bursa pentapeptide (BP5) can promote T to drench
The proliferation of bar cell and bone-marrow-derived lymphocyte, can also improve the anti-mistake of body at the level of the humoral immunity for improving body and cellular immunity
The ability of oxidative stress is the drug of a kind of immunological regulation with wide application prospect, immunization therapy and Hardy dwarfing
Or preparation.It can be used for the fields such as basic research, clinical treatment, nutrition and health care and beauty and make-up.
This patent describe potential use of the bursa pentapeptide in terms of immunization therapy, immunological regulation, but do not refer to
Application in avian influenza vaccine.
Obligee University Of Science and Technology Of He'nan has applied for that Chinese patent CN201310069941.6 discloses recombination T α in 2013
1-BP5 fusogenic peptide, gene, engineering bacteria and application.The recombination fusogenic peptide is that thymosin α1 and bursa pentapeptide BP5 pass through flexibility
Linker is merged.The present invention will recombinate T α 1-BP5 fusion peptide gene insertion expression vector, convert Escherichia coli, obtain high
The genetic engineering bacterium of effect expression recombination T α 1-BP5 fusogenic peptide, recombinates T α 1-BP5 fusogenic peptide as made from Liquid Culture, purifying,
Enterokinase of the fusogenic peptide through N-terminal with His label removes thioredoxin, then through affinitive layer purification, can get single
Recombinate T α 1-BP5 fusogenic peptide.The recombination T α 1-BP5 fusogenic peptide of the invention can be used as the immune assistant of novel polypeptide that cooperation vaccine uses
Agent can effectively enhance Cellular Immunity and humoral immunity level, have broad application prospects.
The invention is to carry out DNA recombinant expression with thymosin α1 on the basis of bursa pentapeptide BP5, has reached immune
The purpose of enhancing.
By above-mentioned record it can be found that BP5 has the potentiality to be exploited of very big Immune-enhancing effect.It researches and develops this field
This aspect carries out in-depth study.
So it is an object of the present invention to being proposed for the new application of BP5 for BP5 progress more in-depth study
Formula, to improve immune effect of vaccine.
Summary of the invention
The purpose of the present invention is to provide a kind of immunopotentiators, meanwhile, the invention also discloses the immunopotentiators
Preparation method, using the avian influenza vaccine of the immunopotentiator and the application in terms of avian influenza vaccine.The immunopotentiator energy
Enough effective antibody titer expressions for improving vaccine immunity.And immunopotentiator preparation is simple, prospects for commercial application
It is good.
Before illustrating the solution of the present invention, give necessary explanation to writing a Chinese character in simplified form:
LB liquid medium: Luria-Bertani fluid nutrient medium；
IPTG: isopropylthiogalactoside, Isopropyl β-D-Thiogalactoside；
SDS-PAGE electrophoresis: polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis；
Ni column: nickel column；
Binding buffer: ni-sepharose purification albumen buffer；
Elution buffer: elution buffer；
M: concentration unit, mol/L；
PET-32a: expression vector, fusion protein type High level prokaryotic expression carrier, market are commercially available；
TE buffer(10mM Tris-HCl；1mM EDTA): TE buffer, Tris-HCl concentration are 10mM, and EDTA is dense
Degree is 1mM；
EcoR I: restriction endonuclease, market are commercially available；
Hind III: restriction endonuclease, market are commercially available；
T4 DNA Ligase:T4 DNA ligase, market are commercially available；
E.coli DH5 α: bacillus coli DH 5 alpha；
RosettaTM (DE3): Rosetta (DE3) E. coli expression strains；
APS: astragalus polyose；
BP5: Cys-Lys-Asp-Val-Tyr；
PBS: phosphate buffered saline solution.
To achieve the goals above, present invention provide the technical scheme that a kind of immunopotentiator, including Cys-Lys-Asp-Val-Tyr and
Astragalus polyose；The weight ratio of the Cys-Lys-Asp-Val-Tyr and astragalus polyose is 1:5~1:20.
It should be understood that the amino acid sequence of Cys-Lys-Asp-Val-Tyr as described herein is amino acid sequence Cys-Lys-Arg-
One or more tandem repetitive sequences of Val-Tyr, preferably 1-6 tandem repetitive sequence；In the specific embodiment of this paper,
Application is 5 tandem repetitive sequences of amino acid sequence Cys-Lys-Arg-Val-Tyr as Cys-Lys-Asp-Val-Tyr used in experiment.
In existing theoretical inference, the number of iterations of repetitive sequence is fewer, is more able to maintain the stable structure of protein,
So in theory, the effect of the Western Immuno enhancing of single Cys-Lys-Asp-Val-Tyr sequence is best.
In above-mentioned immunopotentiator, the weight ratio of the Cys-Lys-Asp-Val-Tyr and astragalus polyose is 1:15.
In above-mentioned immunopotentiator, it is made of following weight percent composition:
Cys-Lys-Asp-Val-Tyr 1%；
Astragalus polyose 15%；
The water of surplus.
Meanwhile the invention also discloses a kind of avian influenza vaccine, Immune-enhancing effect as described above is contained in the vaccine
Agent；The dosage of immunopotentiator is 5-15 μ l/ plumage part in the avian influenza vaccine.
It is all that can play in the amount ranges of the immunopotentiator not less than 5 μ l/ plumage parts by verification experimental verification
Substantive immunoenhancement result.
In above-mentioned avian influenza vaccine, the antigen in the avian influenza vaccine is the inactivation of H9N2 subtype avian influenza virus
Antigen or H5N1 subtype avian influenza virus inactivation antigen.
In addition, the invention also discloses the preparation methods of a kind of as above any immunopotentiator, by Cys-Lys-Asp-Val-Tyr
In water with astragalus polyose mixed dissolution.
In the preparation method of above-mentioned immunopotentiator, include the following steps:
Step 1: constructing the gene of Cys-Lys-Asp-Val-Tyr；
Step 1 specifically: using 5'-TGCAAACGCGTGTAC-3' as BP5 gene, be separately added into EcoR I before and after segment
With III restriction enzyme site of Hind and protectiveness base, the reverse complementary sequence R of fragment sequence F and fragment sequence F are obtained, reversely
Complementary series R and fragment sequence F constitutes the nucleic acid fragment of the gene of Cys-Lys-Asp-Val-Tyr, the fragment length 93bp of fragment sequence F, Bursin
The nucleic acid fragment of the gene of pentapeptide is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Fragment sequence F (specifically refers to sequence table SEQ ID NO:1):
The reverse complementary sequence R (specifically referring to sequence table SEQ ID NO:2) of fragment sequence F:
The sequence of I restriction enzyme site of EcoR is as shown in underscore: GAATTC；
The sequence of III restriction enzyme site of Hind is as shown in underscore: AAGCTT；
Step 2: the gene of Cys-Lys-Asp-Val-Tyr being connected on expression vector pET-32a, recombinant plasmid pET-32a- is obtained
(BP5)5；
Step 2 specifically: take 1 μ g of nucleic acid fragment to be dissolved in 50 μ L TE buffer solutions, 10mM is contained in the buffer solution
Tris-HCl and 1mM EDTA carries out double digestion processing to genetic fragment with EcoR I and III restriction endonuclease of Hind；Meanwhile it also using
EcoR I and III restriction endonuclease of Hind carry out double digestion processing to pET-32a carrier；
Genetic fragment after digestion, pET-32a carrier are mixed, and T4 DNA ligase is added into mixed system and carries out
Connection reaction；Connection obtains connection product after reaction；
Connection product is converted to Escherichia coli reference culture E.coli DH5 α, verifies positive bacterium colony with carrier sequencing primer,
And to positive colony sequencing；The correct recombinant plasmid pET-32a- (BP5) of saving sequence5。
Step 3: by recombinant plasmid pET-32a- (BP5)5It imported into progress inducing expression in Escherichia coli and obtains expression production
Object；
Plasmid imports: taking 5 μ L recombinant plasmid pET-32a- (BP5)5It is added to 100 μ L colibacillus engineerings
In RosettaTM (DE3), it is sufficiently mixed 30min on ice, mixture is as water-bath 90s in 42 DEG C, after taking-up rapidly on ice
3min is stood, plasmid is completed and converts to obtain seed bacterium solution；
Inducing expression: the seed bacterium solution 0.1mL for taking PCR identification positive, the light absorption value of the seed bacterium solution at 600nm wavelength
OD600=0.1, it is inoculated into 5mL LB liquid medium, 37 DEG C of shake cultures to OD600When=0.4~0.6, IPTG is added extremely
Final concentration of 1mM is placed in 25 DEG C of inducing expression 6h.Supernatant precipitating is collected after centrifugation respectively, and is reflected using SDS-PAGE electrophoresis
Fixed, product is present in medium supernatant, based on solubility expression.
Step 4: by expression product by purifying, being drying to obtain Cys-Lys-Asp-Val-Tyr；
Product purification: Ni column is loaded, is added after Binding buffer balance and is slowly added to medium supernatant；To all
It after sample flows through Ni column, is first rinsed with appropriate Binding buffer, removes foreigh protein removing, be slow added into Elution
Buffer collects eluent in peak value；Eluent obtains soluble (BP5) through concentration gradient dialysis renaturation5Thioredoxin,
Size about 22KDa；Enterokinase processing is added to purified product, cuts off thioredoxin, obtains (BP5)5Fusogenic peptide；Again to upper
State product (BP5)5Fusogenic peptide carries out Ni column chromatography and dialysis renaturation, obtains high-purity (BP5)5Fusogenic peptide；Product frozen dried is
For Cys-Lys-Asp-Val-Tyr used in the present invention.
Step 5: by Cys-Lys-Asp-Val-Tyr and astragalus polyose mixed dissolution in water for injection.
(BP5) that the above method is prepared5The amino acid sequence of fusogenic peptide is (to specifically refer to sequence table SEQ ID
NO:3):
COOH-E-F-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-C-K-R-V-Y-K-L-NH2；
That is: COOH-E-F- (C-K-R-V-Y) 5-K-L-NH2
Repetitive sequence is C-K-R-V-Y.
Finally, being used as avian influenza vaccine the invention also discloses the application of a kind of as above any immunopotentiator
Immunity enhancement adjuvant.
The beneficial effects of the present invention are:
The present invention is used cooperatively using Cys-Lys-Asp-Val-Tyr and astragalus polyose, synergistic effect is played in effect, with avian influenza vaccine
Compatibility can get ideal immune effect.
Specifically, using the avian influenza vaccine of immunopotentiator of the invention within the entire immune period, compared to not having
There is the avian influenza vaccine of addition immunopotentiator, the antibody titer of Immune expression can high 3 titres (log2).
Detailed description of the invention
Fig. 1 is the antibody level table that H9N2 subtype avian influenza vaccine immunity of the invention is tested；
Fig. 2 is the antibody level table that H5N1 subtype avian influenza vaccine immunity of the invention is tested；
Fig. 3 is the antibody level table that H9N2 subtype avian influenza vaccine of the invention adds Cys-Lys-Asp-Val-Tyr immunity test；
Fig. 4 is the antibody level table that H9N2 subtype avian influenza vaccine of the invention adds astragalus polyose immunity test；
Fig. 5 is recombination (BP5)5The SDS-PAGE electrophoresis of expression of the fusogenic peptide in Escherichia coli.
Specific embodiment
Illustrate the present invention With reference to embodiment: but it is understood that these specific embodiments are only intended to
Illustrate the present invention, rather than limiting the invention.Those skilled in the art completely can under the inspiration of the present invention, to this
The specific embodiment or technical characteristic of invention improve, but these still belong to by the technical solution improved or replaced
In protection scope of the present invention.
Embodiment one
H9N2 subtype avian influenza vaccine
The component of 500 plumage part vaccines specifically includes:
3 parts of oily phases (90ml), 2 parts of water phases (60ml)；
It is oily mutually to contain 84.6ml white oil (EXonMobil company), 5.4ml Si Ben -80 (the super Industrial Co., Ltd. in Zhaoqing)；
Water phase contains 52.6ml H9N2 blastochyle inactivation antigen (107EID50/ 0.1mL, the big magnificent agriculture biologics in Zhaoqing are limited
Company), 2.4ml Tween-80 (the super Industrial Co., Ltd. in Zhaoqing), 5ml immunopotentiator.
Immunopotentiator containing 10 μ l/ plumage parts in water phase, component are as follows: Cys-Lys-Asp-Val-Tyr 1wt%；Astragalus polyose
15wt%；The water of surplus.
Immunopotentiator the preparation method comprises the following steps:
Step 1: constructing the gene of Cys-Lys-Asp-Val-Tyr；
Using 5'-TGCAAACGCGTGTAC-3' as BP5 gene, III digestion of EcoR I and Hind is separately added into before and after segment
Site and protectiveness base, fragment length 93bp, by bioengineering (Shanghai), limited liability company is synthesized.
Fragment sequence F:
Reverse complementary sequence R:
Step 2: the gene of Cys-Lys-Asp-Val-Tyr being connected on expression vector pET-32a, recombinant plasmid pET-32a- is obtained
(BP5)5；
1 μ g of nucleic acid fragment is taken to be dissolved in 50 μ L TE buffer (10mM Tris-HCl；1mM EDTA) in, with I He of EcoR
III enzymic digestion of Hind, identical collagenase treatment pET-32a carrier；Segment after digestion is mixed with carrier, uses T4 DNA
Ligase connection；Connection product is converted to E.coli DH5 α, verifies positive bacterium colony with carrier sequencing primer, and to positive colony
Sequencing；The correct recombinant plasmid pET-32a- (BP5) of saving sequence5。
Step 3: by recombinant plasmid pET-32a- (BP5)5It imported into progress inducing expression in Escherichia coli and obtains expression production
Object；
Plasmid imports: taking 5 μ L recombinant plasmid pET-32a- (BP5)5It is added to 100 μ L colibacillus engineerings
In RosettaTM (DE3), it is sufficiently mixed 30min on ice, mixture is as water-bath 90s in 42 DEG C, after taking-up rapidly on ice
3min is stood, plasmid conversion is completed.
Inducing expression: the seed bacterium solution 0.1mL (OD for taking PCR identification positive600=0.1), it is inoculated into 5mL LB Liquid Culture
In base, 37 DEG C of shake cultures to OD600When=0.4~0.6, IPTG to final concentration of 1mM is added, is placed in 25 DEG C of inducing expression 6h.
Supernatant precipitating is collected after centrifugation respectively, and uses SDS-PAGE electroresis appraisal, product is present in culture supernatant, with soluble table
Based on reaching, Fig. 5 is as a result seen.The meaning of each symbol in Fig. 5 are as follows: M: Protein Marker；1: culture supernatant；2: bacterial sediment；
3: negative control；
Step 4: by expression product by purifying, being drying to obtain Cys-Lys-Asp-Val-Tyr；
Product purification: Ni column is loaded, is added after Binding buffer balance and is slowly added to culture supernatant；To all samples
It after flowing through Ni column, is first rinsed with appropriate Binding buffer, removes foreigh protein removing, be slow added into Elution buffer,
Peak value collects eluent；Eluent obtains solubility BP5- thioredoxin, size about 22KDa through concentration gradient dialysis renaturation；
Enterokinase processing is added to purified product, cuts off thioredoxin, obtains (BP5)5Fusogenic peptide；Ni is carried out to above-mentioned product again
Column chromatography and dialysis renaturation, obtain high-purity (BP5)5Fusogenic peptide；Product frozen dried is Cys-Lys-Asp-Val-Tyr used in the present invention.
Step 5: by Cys-Lys-Asp-Val-Tyr and astragalus polyose mixed dissolution in water for injection.
Following embodiments, comparative example, BP5 is Cys-Lys-Asp-Val-Tyr prepared by the step 4 of the present embodiment used in immunity test.
Embodiment 2
H9N2 subtype avian influenza vaccine, substantially with embodiment 1, immunopotentiator component are as follows: Cys-Lys-Asp-Val-Tyr 1wt%；It is yellow
Astragalus polysaccharides 5wt%；The water of surplus.Immunopotentiator is 15 μ l/ plumage parts in vaccine.
Embodiment 3
H9N2 subtype avian influenza vaccine, substantially with embodiment 1.Immunopotentiator component are as follows: Cys-Lys-Asp-Val-Tyr 1wt%；It is yellow
Astragalus polysaccharides 20wt%；The water of surplus.Immunopotentiator is 5 μ l/ plumage parts in vaccine.
Embodiment 4
H5N1 subtype avian influenza vaccine
The component of 500 plumage part vaccines specifically includes:
3 parts of oily phases (90ml), 2 parts of water phases (60ml)；
It is oily mutually to contain 84.6ml white oil (EXonMobil company), 5.4ml Si Ben -80 (the super Industrial Co., Ltd. in Zhaoqing)；
Water phase contains 52.6ml H5N1 blastochyle inactivation antigen (108EID50The big magnificent agriculture biotechnology of/0.1mL, Guangdong Wen Shi
Co., Ltd), 2.4ml Tween-80 (the super Industrial Co., Ltd. in Zhaoqing), 5ml immunopotentiator.
Immunopotentiator containing 10 μ l/ plumage parts in water phase, component are as follows: Cys-Lys-Asp-Val-Tyr 1wt%；Astragalus polyose
15wt%；The water of surplus.The preparation method is the same as that of Example 1 for immunopotentiator.
Comparative example 1
H9N2 subtype avian influenza vaccine is free of astragalus polyose, component substantially with embodiment 1 in immunopotentiator are as follows:
Cys-Lys-Asp-Val-Tyr 1wt%；The water of surplus.
Comparative example 2
H9N2 subtype avian influenza vaccine, substantially with embodiment 1.Cys-Lys-Asp-Val-Tyr, component are free of in immunopotentiator are as follows:
Its component are as follows: astragalus polyose 15wt%；The water of surplus.
1, H9N2 subtype avian influenza vaccine immunity is tested
1.1 zooperies grouping
40 7 age in days SPF chickens are randomly divided into four groups, every group 10: A: conventional H 9N2 hypotype inactivates avian influenza vaccine, neck
Dorsal sc injection, 0.3ml/ plumage；B: inactivated vaccine+APS-BP5 (embodiment 1)；C:APS-BP5 control group (10 μ l/0.3ml/
Plumage, APS-BP5 levels are with embodiment 1)；D:PBS control group (every immune 0.3ml/ plumage).
The acquisition of 1.2 samples
Before immune and 1st~10 week after immune, venous blood collection 1mL under every chicken wings, 3000rpm are centrifuged 10min points weekly
From serum, -20 DEG C of freezen protectives.
1.3 result
Antibody level measurement.Specific antibody level is detected by hemagglutination inhibition test (HI test) (HI test), such as
Shown in Fig. 1,3 weeks after being immunized, avian influenza virus (H9) antibody level of APS-BP5 vaccine group starts to be significantly higher than conventional vaccine group
(p < 0.01), and it is continued until off-test.To off-test, the antibody water of APS-BP5 vaccine group 3 weeks after immune
It is flat to be consistently higher than 3 titres of conventional vaccine group or more, it is higher than 7 titres of conventional vaccine group, peak value in its antibody level of 4th week
Antibody titer is greater than 11 (log2).
2, H9N2 subtype avian influenza vaccine Cys-Lys-Asp-Val-Tyr adjuvant immunity is tested
2.1 zooperies grouping
40 7 age in days SPF chickens are randomly divided into four groups, every group 10: A: conventional H 9N2 hypotype inactivates avian influenza vaccine, neck
Dorsal sc injection, 0.3ml/ plumage；B: inactivated vaccine+BP5 (comparative example 1)；(10 μ l/0.3ml/ plumages, BP5 are dense for C:BP5 control group
Content is spent with comparative example 1)；D:PBS control group (every immune 0.3ml/ plumage).
The acquisition of 2.2 samples
Before immune and 1st~10 week after immune, venous blood collection 1mL under every chicken wings, 3000rpm are centrifuged 10min points weekly
From serum, -20 DEG C of freezen protectives.
2.3 result
Antibody level measurement.Specific antibody level is detected by hemagglutination inhibition test (HI test) (HI test), such as
Shown in Fig. 2,2 weeks after being immunized, avian influenza virus (H9) antibody level of Cys-Lys-Asp-Val-Tyr Adjuvanted vaccines group starts gradually to be higher than conventional
Vaccine group (p < 0.01), and it is continued until off-test.To off-test, Cys-Lys-Asp-Val-Tyr vaccine group 3 weeks after immune
Antibody level be consistently higher than about 2 titres of conventional vaccine group.
3, H9N2 subtype avian influenza vaccine astragalus polysaccharide adjuvant immunity test
3.1 zooperies grouping
40 7 age in days SPF chickens are randomly divided into four groups, every group 10: A: conventional H 9N2 hypotype inactivates avian influenza vaccine, neck
Dorsal sc injection, 0.3ml/ plumage；B: inactivated vaccine+APS (comparative example 2)；C:APS control group (10μl/ 0.3ml/ plumage, APS are dense
Content is spent with comparative example 2)；D:PBS control group (every immune 0.3ml/ plumage).
The acquisition of 3.2 samples
Before immune and 1st~10 week after immune, venous blood collection 1mL under every chicken wings, 3000rpm are centrifuged 10min points weekly
From serum, -20 DEG C of freezen protectives.
3.3 result
Antibody level measurement.Specific antibody level is detected by hemagglutination inhibition test (HI test) (HI test), such as
Shown in Fig. 3, after being immunized, avian influenza virus (H9) antibody level of astragalus polysaccharide adjuvant vaccine group is slightly above conventional vaccine group, energy
It is continued until off-test.
4, H5N1 subtype avian influenza vaccine immunity is tested
4.1 zooperies grouping
40 7 age in days SPF chickens are randomly divided into four groups, every group 10: A: conventional H 5N1 hypotype inactivates avian influenza vaccine, neck
Dorsal sc injection, 0.3ml/ plumage；B:H5N1 inactivated vaccine+APS-BP5 (embodiment 4)；C:APS-BP5 control group (10 μ l/
0.3ml/ plumage, APS-BP5 levels are with embodiment 4)；D:PBS control group (every immune 0.3ml/ plumage)
The acquisition of 4.2 samples
Before immune and 1st~10 week after immune, venous blood collection 1mL under every chicken wings, 3000rpm are centrifuged 10min points weekly
From serum, -20 DEG C of freezen protectives.
4.3 result
Antibody level measurement.Specific antibody level is detected by hemagglutination inhibition test (HI test) (HI test), such as
Shown in Fig. 4,2 weeks after being immunized, avian influenza virus (H5) antibody level of APS-BP5 vaccine group starts to be significantly higher than conventional vaccine group
(p < 0.01), and it is continued until off-test.To off-test, the antibody water of APS-BP5 vaccine group 3 weeks after immune
It is flat to be consistently higher than 3 titres of conventional vaccine group or more.
By above-mentioned immunity test, we are available to draw a conclusion:
Bursin (BP) is the small molecule skin substance being present in bird bursal, has substantial connection with immune function.Capsule
Plain pentapeptide (BP5) is one of Bursin family, has induction poultry bone-marrow-derived lymphocyte precursor differentiation, proliferation, improves antibody water
It is flat, enhance humoral immunity；The activity of conversion for promoting T lymphocyte, to enhance the effect of cellular immunity.
Polysaccharide compound is a kind of good immunomodulator, the effect in terms of improving and improving Immune Function In Animals
Increasingly attract people's attention.Astragalus polyose is main bioactive ingredients in Radix Astragali root extract.Studies have shown that Radix Astragali
Polysaccharide energy stimulating expression of macrophage activity, promotes T cell proliferation, moreover it is possible to promote expression of the surface antigen in lymphocyte, induce
Stronger humoral and cellular immune response response plays a significant role in non-specific and specific immune response.In general, yellow
Astragalus polysaccharides have immunological regulation, improve immune response, alleviate the multiple functions such as immunologic function disorder caused by environmental stress.
Being used in combination for two kinds of immunopotentiators, plays synergistic effect in effect, can get reason with avian influenza vaccine compatibility
The immune effect thought.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>ZhaoQing DaHuaNong Biological medicine Co., Ltd
Co., Ltd, Hua Nong (Zhaoqing) biological industry Institute for Research and Technology
<120>a kind of immunopotentiator, preparation method, avian influenza vaccine and application
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 93
<212> DNA
<213>artificial sequence ()
<400> 1
agcgaattct gcaaacgcgt gtactgcaaa cgcgtgtact gcaaacgcgt gtactgcaaa 60
cgcgtgtact gcaaacgcgt gtacaagctt cac 93
<210> 2
<211> 93
<212> DNA
<213>artificial sequence ()
<400> 2
gtgaagcttg tacacgcgtt tgcagtacac gcgtttgcag tacacgcgtt tgcagtacac 60
gcgtttgcag tacacgcgtt tgcagaattc gct 93
<210> 3
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<212> PRT
<213>artificial sequence ()
<400> 3
Glu Phe Cys Lys Arg Val Tyr Cys Lys Arg Val Tyr Cys Lys Arg Val
1 5 10 15
Tyr Cys Lys Arg Val Tyr Cys Lys Arg Val Tyr Lys Leu
20 25
Claims (10)
1. a kind of immunopotentiator, it is characterised in that: including Cys-Lys-Asp-Val-Tyr and astragalus polyose；The Cys-Lys-Asp-Val-Tyr and Radix Astragali is more
The weight ratio of sugar is 1:5~1:20.
2. immunopotentiator according to claim 1, it is characterised in that: the weight of the Cys-Lys-Asp-Val-Tyr and astragalus polyose
Than for 1:15.
3. immunopotentiator according to claim 1 or 2, it is characterised in that: be made of following weight percent composition:
Cys-Lys-Asp-Val-Tyr 1%；
Astragalus polyose 15%；
The water of surplus.
4. a kind of avian influenza vaccine, it is characterised in that: contain immune increasing a method according to any one of claims 1-3 in the vaccine
Strong agent；The dosage of immunopotentiator is 5-15 μ l/ plumage part in the avian influenza vaccine.
5. avian influenza vaccine according to claim 4, it is characterised in that: the antigen in the avian influenza vaccine is H9N2
Subtype avian influenza virus inactivation antigen or H5N1 subtype avian influenza virus inactivation antigen.
6. a kind of preparation method of the immunopotentiator as described in claims 1 to 3 is any, it is characterised in that: by Cys-Lys-Asp-Val-Tyr
In water with astragalus polyose mixed dissolution.
7. the preparation method of immunopotentiator according to claim 6, which comprises the steps of:
Step 1: constructing the gene of Cys-Lys-Asp-Val-Tyr；
Step 2: the gene of Cys-Lys-Asp-Val-Tyr being connected on expression vector pET-32a, recombinant plasmid pET-32a- (BP5) is obtained5；
Step 3: by recombinant plasmid pET-32a- (BP5)5It imported into progress inducing expression in Escherichia coli and obtains expression product；
Step 4: by expression product by purifying, being drying to obtain Cys-Lys-Asp-Val-Tyr；
Step 5: by Cys-Lys-Asp-Val-Tyr and astragalus polyose mixed dissolution in water for injection.
8. the preparation method of immunopotentiator according to claim 7, which is characterized in that the step 1 and step 2 tool
Body are as follows:
Step 1: using 5'-TGCAAACGCGTGTAC-3' as BP5 gene, being separately added into III enzyme of EcoR I and Hind before and after segment
Enzyme site and protectiveness base obtain fragment sequence F, fragment length 93bp, by Sangon Biotech (Shanghai) Co., Ltd.
It synthesizes and provides；
Step 2: taking 1 μ g of nucleic acid fragment to be dissolved in 50 μ L TE buffer buffer solutions, obtained with EcoR I and III enzymic digestion of Hind
Segment after digestion；
With III collagenase treatment pET-32a carrier of EcoR I and Hind；
Segment after digestion is mixed with the pET-32a carrier after collagenase treatment, after T4 DNA Ligase connection digestion
Segment and pET-32a carrier obtain connection product；
Connection product is converted to E.coli DH5 α, verifies positive bacterium colony with carrier sequencing primer, and to positive colony sequencing；It protects
Deposit the correct recombinant plasmid pET-32a- (BP5) of sequence5。
9. the preparation method of immunopotentiator according to claim 8, which is characterized in that the step 3 specifically:
Plasmid imports: taking recombinant plasmid pET-32a- (BP5)5It is added in Escherichia coli RosettaTM (DE3), on ice sufficiently
Mixing, mixture are stood rapidly after taking-up on ice as water-bath in 42 DEG C, are completed plasmid conversion, are obtained seed bacterium solution；
Inducing expression: the seed bacterium solution for taking PCR identification positive is inoculated into culture medium, culture to OD600When=0.4~0.6, it is added
IPTG, in 25 DEG C of progress inducing expressions；Identified, product is present in medium supernatant, based on solubility expression.
10. a kind of application of the immunopotentiator as described in claims 1 to 3 is any, which is characterized in that be used as avian influenza vaccine
Immunity enhancement adjuvant.
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| CN111544587A (en) * | 2020-06-29 | 2020-08-18 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and application thereof |
| CN111603558A (en) * | 2020-06-29 | 2020-09-01 | 肇庆大华农生物药品有限公司 | Immunopotentiator and application thereof in avian influenza vaccine |
| WO2022000205A1 (en) * | 2020-06-29 | 2022-01-06 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and use thereof |
| CN116813795A (en) * | 2023-05-25 | 2023-09-29 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
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| CN101255189A (en) * | 2008-04-07 | 2008-09-03 | 广东大华农动物保健品有限公司 | Animal vaccine immunopotentiator and production method thereof |
| CN101434650A (en) * | 2008-12-23 | 2009-05-20 | 李德元 | Bursa pentapeptide, deriving peptide thereof and use thereof |
| CN102000332A (en) * | 2010-11-16 | 2011-04-06 | 南京农业大学 | Composite adjuvant for duck bird flu oral mucosal immunization |
| CN103145854A (en) * | 2013-03-05 | 2013-06-12 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
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| CN101434650A (en) * | 2008-12-23 | 2009-05-20 | 李德元 | Bursa pentapeptide, deriving peptide thereof and use thereof |
| CN102000332A (en) * | 2010-11-16 | 2011-04-06 | 南京农业大学 | Composite adjuvant for duck bird flu oral mucosal immunization |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111544587A (en) * | 2020-06-29 | 2020-08-18 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and application thereof |
| CN111603558A (en) * | 2020-06-29 | 2020-09-01 | 肇庆大华农生物药品有限公司 | Immunopotentiator and application thereof in avian influenza vaccine |
| WO2022000205A1 (en) * | 2020-06-29 | 2022-01-06 | 肇庆大华农生物药品有限公司 | Avian influenza vaccine adjuvant and use thereof |
| CN116813795A (en) * | 2023-05-25 | 2023-09-29 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
| CN116813795B (en) * | 2023-05-25 | 2024-01-30 | 华中农业大学 | Recombinant AaLS-BSP fusion peptide, preparation method and application |
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| WO2020215351A1 (en) | 2020-10-29 |
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